Cyclopeptolides

ABSTRACT

Cyclopeptolides of formula (I), wherein A, B, R 1  Leu, Leu, C, X and Y are as defined, are inhibitors of adhesion molecule, expression and inhibitors of TNF release and are thus useful for treatment of inflammatory and other diseases which involve increased levels of adhesion molecule expression and/or are mediated by TNF. ##STR1##

This ia a 371 of PCT/EP/96/05123, filed Nov. 20, 1996.

This invention relates to cyclopeptolides and to their therapeutic useas inhibitors of adhesion molecule expression.

Cellular adhesion molecules such as ICAM-1, VCAM-1 and E-selectin areexpressed on the surface of endothelial cells, as well as keratinocytesfor ICAM-1, in response to pro-inflammatory mediators including TNFα,IFNγ, IL1 and LPS. Corresponding counter-ligands, e.g. LFA-1, VLA4 andSLE^(x), are expressed on the surfaces of circulating blood cells.Transendothelial migration of leucocytes during inflammatory processes,as well as extravascular cell-cell interactions, are regulated as aresult of the interactions between these adhesion molecules and theircounter-ligands. Consequently, inhibitors of adhesion moleculeexpression offer potential for the treatment of many disease states.

Cyclopeptolides are cyclic molecules comprising amino acid residueslinked together by peptide bonds and at least one hydroxy substitutedcarboxylic acid residue which is linked through its hydroxyl substituentto the neighbouring acid residue by an ester linkage.

Our copending patent application, published International patentapplication WO 96/03430 discloses novel cycloheptapeptolides which areinhibitors of ICAM-1, VCAM-1 and E-selectin expression. We have nowdiscovered further new cycloheptapeptolides of the same general compoundclass, including compounds having particularly desirable properties.

The present invention provides cycloheptapeptolides of formula I##STR2## wherein: A is a glycolic acid residue optionally α-substitutedby

H, methyl, ethyl, propyl or vinyl, optionally substituted by

halogen, alkoxy, optionally protected hydroxy or amino, CSNH₂, COOR₂,vinyl,

--C.tbd.CH or thiazole,

wherein R₂ is H or lower alkyl,

optionally substituted by

alkyl, halogen, cycloalkyl, optionally substituted thiazole, COOR₂ or

--C.tbd.CH,

wherein R₂ is as defined above;

B is an α-amino-γ-methyl-substituted octanoic acid residue;

R₁ is hydrogen or methyl;

C is a tryptophan or N-methyl-tryptophan residue of formula II ##STR3##wherein R₃ represents hydrogen, alkoxy, alkyl or benzyl, R₄ representshydrogen or

halogen, R₅ represents hydrogen or methyl and ═ represents a single ordouble bond;

X is an α-amino-substituted (C₂ to C₁₄) carboxylic acid residue, and

Y is an α-amino- or N-methyl-α-amino substituted (C₂ to C₁₀) carboxylicacid residue.

In formula I the N-terminal to C-terminal orientation of the amino acidresidues is in the clockwise direction, and the peptolide ester bond isbetween residues A and Y. When R₁ is methyl, the residues R₁ -Leu andLeu are N-methyl-leucine and leucine residues respectively.

Preferably A is a glycolic acid residue, which is α-substituted by H,methyl, ethyl or propyl optionally substituted by amino, hydroxy,chloro, alkoxy, optionally substituted thiazole, optionally substitutedvinyl, cyclopropyl, CSNH₂ or --C.tbd.CH.

Preferably C is a N-methyltryptophan residue of formula II, wherein R₃represents hydrogen, (C₁ to C₄)alkoxy (especially methoxy) or alkyl andR₄ represents hydrogen or halogen.

Preferably X is an ox-amino-substituted (C₄ to C₈) carboxylic acidresidue, which is optionally β- or γ-(C₁ to C₄) alkyl substituted. Mostpreferably X is an α-amino-β- or γ-(C₁ to C₄) alkyl-, especiallymethyl-, substituted octanoic or a butyric acid residue.

Preferably Y is an N-methyl-α-amino-substituted (C₂ to C₄) carboxylicacid residue, which is optionally β- or γ-(C₁ to C₄) alkyl-substituted.Most preferably Y is an N-methyl-alanine or N-methyl-valine residue.

The invention includes open chain peptides or peptolides correspondingto the compounds of formula I; for instance, the open chain moleculesobtained by either cleavage of the ester bond between residues Y and Aor cleavage of an amide linkage between any other adjacent pair of theacid residues. Preferred open-chain derivatives are compounds offormulae IV and V

    H--C--X--Y--A--B--R.sub.1 Leu--Leu.OR.sub.7                IV

    and

    HA--B--R.sub.1 Leu--Leu--C--X--Y.OR.sub.7                  V

wherein R₇ represents hydrogen or alkyl, e.g. C₁₋₄ lower alkyl.

Preferred compounds according to one embodiment of the invention are thecompounds of formula Ip ##STR4## wherein: A_(p) is a glycolic acidresidue optionally α-substituted by H, ethyl or methyl;

B_(p) is an α-amino-γ-methyl-substituted octanoic acid residue;

R_(1p) is hydrogen or methyl;

C_(p) is a tryptophan or N-methyl-tryptophan residue, which isoptionally N'-(C₁ to C₄) alkoxy substituted;

X_(p) is an α-amino-substituted (C₂ to C₁₄) carboxylic acid residue, and

Y_(p) is an α-amino- or N-methyl-α-amino substituted (C₂ to C₁₀)carboxylic acid residue.

Preferred compounds according to a further embodiment of the inventionare compounds of formula I_(p) ' ##STR5## wherein B_(p), R_(1p), C_(p),X_(p) and Y_(p) are as defined above and A'p is an α hydroxy-substitutedbutyric acid residue which is γ substituted by a group of formula VI##STR6## wherein R₂ represents a lower alkyl group e.g. a C₁₋₄ loweralkyl group.

Most preferably R₂ is methyl or ethyl.

Preferred compounds according to a yet further embodiment of theinvention are compounds of formula I_(p) " ##STR7## wherein B_(p),R_(1p), C_(p), X_(p) and Y_(p) are as defined above and A_(p) " is an ahydroxy substituted butyric acid residue γ substituted by a group offormula VII ##STR8## wherein R₆ represents hydrogen, lower alkyl orphenyl or forms a carbocyclic ring together with position 5 of thethiazolyl ring.

Compounds of formula I, IV, V, Ip, Ip' and Ip" are hereinafter referredto as "compounds of the invention", which term also includes all thecompounds of the invention when in salt or ester form as well as in freeform.

The compounds of the invention contain asymmetric atoms and thus mayexist in a number of epimeric forms. All of the possible epimers as wellas diastereoisomeric mixtures thereof are encompassed in the invention.Epimers which possess inhibition of adhesion molecule expressionactivity are preferred. In general, e.g. for pharmaceutical use inaccordance with the invention, epimers which possess inhibition ofadhesion molecule expression activity in pure or substantially pure form(i.e. free or substantially free of epimers which lack inhibition ofadhesion molecule expression activity ), e.g. comprising at least 90%,e.g. at least 95% of active epimer (i.e. comprising less than 10%, e.g.less than 5% of inactive epimer) will be preferred. Most preferablycompounds of the invention have the same cyclopeptolide ringstereochemical conformation as the particularly preferred compound offormula VII below.

Particularly preferred compounds of the invention are the compounds offormulae VIII, IX and X ##STR9##

The compound of formula VIII has been isolated from cultures of fungalstrain F/94-499709, samples of which were deposited with theDSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen under theprovisions of the Budapest Treaty on Sep. 18, 1995 and are identified asdeposit number DSM 10275. The characteristics of fungal strainF/94-499709 are described hereinafter in Example 1. The compound offormula VIII is a particular compound of the invention.

Samples of strain F/94-499709 may also be obtained from Sandoz Ltd.CH-4002 Basel Switzerland.

Notice is hereby given that access to samples of DSM 10275 is limited inaccordance with the provisions of Rule 28 (4) and (5) EPC.

The invention includes the strain F/94-499709 (DSM 10275) in isolatedform and mutants and derivatives thereof as well as all novelcyclopeptolides which are produced by this strain.

The compound of formula VII and related compounds may be obtained bycultivating F/94-499709 (DSM 10275) or a mutant or derivative thereof orsimilar fungal species in nutrient medium and recovering the compoundstherefrom, for example as described in Example 2.

The characteristics of the compound of formula VIII are given in Example3.

Compounds according to the invention may be prepared by derivatisationof the compounds of formulae XI or XII (as hereinafter described) orVIII, which comprises

a) for the preparation of compounds of formula I, wherein A issubstituted by COOR₂, reacting corresponding compounds of formula I,wherein A is substituted by CN, with nucleophiles, preferably analcohol, with appropriate basic or acidic catalysis, preferablyhydrochloric acid, in organic solvents, preferably ether, or

b) for the preparation of compounds of formula I, wherein A isalkoxymethyl substituted, reacting corresponding compounds of formula I,wherein A is substituted by CH₂ --OH, with alkylating compounds, such asalkylhalogenides or diazo-compounds with or without catalysts, or

c) for the preparation of compounds of formula I, wherein A issubstituted by COOR₂, esterifying corresponding compounds of formula I,wherein A is substituted bag COOH, by standard methods, preferably byconversion into the acid chloride with e.g. thionyl chloride andtreatment with an appropriate alcohol in the presence or absence of anacid binder, or

d) for the preparation of compounds of formula I, wherein A issubstituted by CH₂ OH, reducing corresponding compounds of formula I,wherein A is substituted by COOR₂, with metal hydrides or boronhydrides, preferably borane dimethylsulfide complex, in organicsolvents, or

e) for the preparation of compounds of formula I, wherein A issubstituted by optionally substituted vinyl, reacting correspondingcompounds of formula I, wherein A is substituted by CHO, with a Wittigreagent, or

f) for the preparation of compounds of formula I, wherein A issubstituted by CH₂ NH₂, reducing corresponding compounds of formula I,wherein A is substituted by CH₂ N₃, or

g) for the preparation of compounds of formula I, wherein A issubstituted by C.tbd.CH, dehydrogenating corresponding compounds offormula I, wherein A is substituted by CH═CBr₂, or

h) for the preparation of compounds of formula I, wherein A issubstituted by cyclopropyl, reacting corresponding compounds of formulaI, wherein A is substituted by vinyl, with diazomethane, or

i) for the preparation of compounds of formula I, wherein A issubstituted by CSNH₂, reacting corresponding compounds of formula Iwherein A is substituted by CN with sulfur derivatives, preferably withdiphenylphosphinodithioic acid, e.g. by refluxing a solution of thesulphur compound with a compound of formula I wherein A is substitutedby CN, or

j) for the preparation of preferred compounds of formula I_(p) "##STR10## wherein the substituents are as defined above, reacting acompound of formula I_(p) " wherein A_(p) " represents ana-hydroxy-substituted butyric acid residue γ-substituted by --CS--NH₂,with a α-halogencarbonyl compound of formula XIII

    Hal--CH.sub.2 --CO--R.sub.6                                XIII

wherein R₆ is as defined above and Hal represents halogen, or with theacetal of the compound of formula XIII

(The reaction may be carried out according to known methods, e.g.reacting a solution of a compound of formula II in a solvent inert underreaction conditions, e.g. in dimethylformamide or pyridine, at elevatedtemperature, preferably at 60° to 100° C. The end products may beisolated and purified by conventional techniques.), or

k) for the preparation of preferred compounds of formula I_(p) '##STR11## wherein the substituents are as defined above, reacting acompound of formula Ip' wherein A_(p) ' represents ana-hydroxy-substituted butyric acid residue which is y-substituted by--CHO, with alkoxycarbonylmethylenetriphenylphosphorane and isolatingthe compounds of formula I_(p) ', or

l) for the preparation of compounds of formula I, wherein R₃ representshydrogen, removing the methoxy group from compounds of formula I,wherein R₃ represents OCH₃, or

m) for the preparation of compounds of formula I, wherein the symbol ═represents a single bond, reducing compounds of formula I, wherein thesymbol ═ represents a double bond, or

n) for the preparation of compounds of formula L wherein R₃ representsalkyl or benzyl, introducing these groups into compounds of formula I,wherein R₃ represents hydrogen, or

o) for the preparation of compounds of formula I, wherein R₄ representshalogen, halogenating compounds of formula I, wherein R₄ representshydrogen, or

p) for the preparation of compounds of formula I, wherein R₃ representsalkoxy and the symbol ═ represents a double bond, reacting compounds offormula I, wherein R₃ represents hydrogen and the symbol ═ represents asingle bond, with an alkali tungstate and hydrogen peroxide andalkylating the N-hydroxy-indol-intermediate, and if desired isolatingthe compound of formula I.

In preferred embodiments of a) to i) above the compounds of formula Iare compounds wherein A is an α-hydroxy butyric acid residue which isγ-substituted by COOR₂, CN, alkoxymethyl, CH₂ --OH, COOH, optionallysubstituted vinyl, CHO, CH₂ NH₂, CH₂ N₃, C.tbd.CH, CH═CBr₂, cyclopropylor vinyl as appropriate.

Intermediates for preparation of compounds of formula I may be preparedas follows:

(i) for preparation of intermediates wherein A is substituted by --CHO,oxidising corresponding compounds of formula I wherein A is substitutedby --CH₂ OH, and

(ii) for preparation of intermediates wherein A is substituted by--COOH, hydrolysing corresponding compounds of formula I wherein A issubstituted by COOAlkyl, with mineral acid e.g. HCl in aqueous alcoholicsolution, or with base.

Intermediates, for preparation of compounds of formula I, wherein A issubstituted by --CN include natural compounds. For example compounds offormula XI and XII. ##STR12## are obtainable as isolates from culturesof fungal strain F92-4471/08, deposited with the US Department ofAgriculture, NRRL culture collection under the provisions of theBudapest Treaty on Jul. 2, 1993 and identified as deposit number NRRL21123. The characteristics of fungal strain F92-4471/08 and theisolation of compounds XI and XII are described in detail in ourcopending patent application, International patent application WO96/03430.

The compounds of the invention may be prepared also by chemicalsynthesis; for example, using conventional peptide synthesis techniques.Typically the final step in the preparation of the compounds is acyclisation step in which a linear peptide or peptolide comprising theacid residues A, B, R₁ Leu, Leu, C, X and Y linked together inappropriate order is cyclised by an amide- or ester-bond formingreaction.

Thus the invention includes a process for the preparation of a cyclicpeptolide of formula I comprising cyclisation of a linear peptide orpeptolide comprising the acid residues A, B, R₁ Leu, Leu, C, X and Ylinked together in appropriate order.

The compounds of the invention exhibit pharmacological activity and aretherefore useful as pharmaceuticals. In particular the compounds of theinvention are inhibitors of the stimulated expression of cellularadhesion molecules, especially inhibitors of VCAM-1 relative toE-selectin and ICAM-1 expression. In particular also the compounds ofthe invention are inhibitors of the release of TNF, e.g. inhibitors ofthe release of TNFα.

Assays which may be used to detect the inhibition of ICAM-1, VCAM-1 andE-selectin expression and the inhibition of TNFα release by thecompounds of the invention are described after the Examples.

Thus, in view of their activity as inhibitors of cellular adhesionmolecule expression, the compounds are useful for the treatment orprophylaxis of disease processes which involve expression of cellularadhesion molecules. These disease processes include many acquired andinherited diseases/disorders where leucocyte activation and traffickingplay a prominent role in the pathogenic process, most notably acute andchronic inflammation (e.g. allergy, asthma, dermatitis, psoriasis,reperfusion injury and septic shock), autoimmune states (e.g. diabetes,multiple sclerosis and rheumatoid arthritis) and immune-mediatedneurodegeneration (e.g. acquired immunodeficiency disorders). Otherindications for the compounds of the invention include tumour metastasis(e.g. melanoma, osteocarcinoma), atherosclerosis and allograft/xenograftrejection, since it is known that inhibition of vascular adhesionmolecules can greatly improve the prognosis of these processes.

Also the compounds of the invention have therapeutic potential inhyperproliferative skin diseases (e.g. psoriasis) as well as variousmalignancies in view of their inhibitory activity at submicromolarconcentrations when tested for 72 hours in a keratinocyte-based as wellas other proliferation assays.

The compounds of the invention are active in inhibitingTNFα/IL-6-induced HIV production in the U1 monocytic cell line, asevaluated by p 24 ELISA and are therefore useful in the treatment ofimmunodeficiencies and virally caused diseases, especially in thetreatment of AIDS.

Furthermore, in view of their activity as inhibitors of TNF release, thecompounds of the invention are useful for the prophylaxis or treatmentof diseases or pathological conditions mediated by TNF, especially TNFα,e.g., inflammatory conditions, autoimmune diseases, severe infections,and organ or tissue transplant rejection including both allograft andxenograft rejection, e.g. for the treatment of recipients of heart,lung, combined heart-lung, liver, kidney, pancreatic, skin or cornealtransplants and for the prevention of graft-versus-host disease, such asfollowing bone marrow transplants.

The compounds of the invention are particularly useful for thetreatment, prevention, or amelioration of autoimmune disease and ofinflammatory conditions, in particular inflammatory conditions with anaetiology including an autoimmune component such as arthritis (forexample rheumatoid arthritis, arthritis chronica progrediente andarthritis deformans) and rheumatic diseases. Specific auto-immunediseases for which the compounds of the invention may be employedinclude autoimmune haematological disorders (including e.g. hemolyticanaemia, aplastic anaemia, pure red cell anaemia and idiopathicthrombocytopenia), systemic lupus erythematosus, polychondritis,sclerodoma, Wegener granulamatosis, dermatomyositis, chronic activehepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome,idio-pathic sprue, autoimmune inflammatory bowel disease (including e.g.ulcerative colitis and Crohn's disease), endocrine ophthalmopathy,Graves disease, sarcoidosis, multiple sclerosis, primary biliarycirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis(anterior and posterior), keratoconjunctivitis sicca and vernalkeratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritisand glomerulonephritis (with and without nephrotic syndrome, e.g.including idiopathic nephrotic syndrome or minimal change nephropathy).

The compounds of the invention are also useful for the treatment,prevention, or amelioration of asthma, bronchitis, pneumoconiosis,pulmonary emphysema, and other obstructive or inflammatory diseases ofthe airways.

The compounds of the invention are useful for treating undesirable acuteand hyperacute inflammatory reactions which are mediated by TNF,especially by TNFα, e.g., acute infections, for example septic shock(e.g., endotoxic shock and adult respiratory distress syndrome),meningitis, pneumonia; and severe burns; and for the treatment ofcachexia or wasting syndrome associated with morbid TNF release,consequent to infection, cancer, or organ dysfunction, especiallyAIDS-related cachexia, e.g., associated with or consequential to HIVinfection.

Thus the invention also includes the therapeutic use of, and therapeuticcompositions containing, the compounds of the invention.

In particular the invention includes methods for the treatment orprophylaxis of diseases which involve expression of adhesion moleculeswhich comprise administering a therapeutically or prophylacticallyeffective amount of a compound according to the invention to a subject.

The invention also includes therapeutic compositions comprising atherapeutically effective amount of a compound according to theinvention.

Furthermore the invention includes the use of a compound according tothe invention for the preparation of a medicament for application in thetreatment or prophylaxis of diseases which involve expression ofadhesion molecules.

In particular the invention also provides in a further series ofembodiments:

A. A method of inhibiting production of soluble TNF, especially TNFα, orof reducing inflammation in a subject (i.e., a mammal, especially ahuman) in need of such treatment which method comprises administering tosaid subject an effective amount of a compound of the invention, or amethod of treating any of the above mentioned conditions, particularly amethod of treating an inflammatory or autoimmune disease or condition,e.g., multiple sclerosis or rheumatoid arthritis, or alleviating one ormore symptoms of any of the above mentioned conditions.

B. A compound of the invention for use as a pharmaceutical, e.g. for usein the prophylaxis or treatment of diseases or pathological conditionsmediated by TNF, e.g. as an immunosuppressant or antiinflammatory agentor for use in the prevention, amelioration or treatment of any diseaseor condition as described above, e.g., an autoimmune or inflammatorydisease or condition.

C. A pharmaceutical composition comprising a compound of the inventionin association with a pharmaceutically acceptable diluent or carrier,e.g., for use in the prophylaxis or treatment of diseases orpathological conditions mediated by TNF, e.g. as an immunosuppressant oranti-inflammatory agent or for use in the prevention, amelioration ortreatment of any disease or condition as described above, e.g., anautoimmune or inflammatory disease or condition.

D. Use of a compound of the invention in the manufacture of a medicamentfor use in the prophylaxis or treatment of diseases or pathologicalconditions mediated by TNF, e.g. as an immunosuppressant oranti-inflammatory agent or for use in the prevention, amelioration ortreatment of any disease or condition as described above, e.g., anautoimmune of inflammatory disease or condition.

The compositions may be for parenteral, oral, aerosol, inhalation ortopical use and usually comprise one or more pharmaceutically acceptablecarriers diluents or excipients and may comprise additives such asstabilisers and the like.

The dosages of the compounds used may be varied having regard to thecondition or disease involved, whether the use is for treatment orprophylaxis thereof and the mode and route of administration among otherthings. In general, however satisfactory results are obtained onadministration orally at dosages of from about 0.05 to about 10mg/kg/day, preferably from about 0.1 to about 7.5 mg/kg/day, morepreferably from about 0.1 to about 2 mg/kg/day administered once or, individed doses, 2 to 4 times per day. Alternatively for parenteraladministration, e.g. by iv drip or infusion, dosages from about 0.01 toabout 5 mg/kg/day, preferably from about 0.05 to about 1 mg/kg/day andmore preferably from about 0.1 to about 1.0 mg/kg/day may be used.Suitable daily dosages for human patients are thus from about 2.5 toabout 500 mg p.o., preferably from about 5 to about 250 mg p.o., morepreferably from about 5 to about 100 mg p.o.; or from about 0.5 to about250 mg i.v., preferably from about 2.5 to about 125 mg i.v. and morepreferably from about 2.5 to about 50 mg i.v.

The compounds may be administered by any appropriate route, includingenterally, parenterally and topically or by inhaler. Suitable enterallyadministered forms ire solutions for drinking, tablets or capsules.Suitable parenteral forms are injectable solutions or suspensions.Suitable forms for topical administration include creams, lotions andthe like at a concentration range of 0.01-10%, preferably from 0.1 to1%, by weight for such formulations. Suitable unit dosage forms for oraladministration may comprise from 1 to 50 mg of the compound, usuallyfrom 1 to 10 mg.

The invention is further described, by way of illustration only, in thefollowing examples which refer to the accompanying diagrams in which:

FIG. 1 shows the UV spectra of the compound of formula VIII;

FIG. 2 shows the IR spectrum of the compound of formula VIII;

FIG. 3 shows the FD-Mass spectrum of the compound of formula VIII;

FIG. 4 shows the FD-Mass spectrum (with addition of LiI) of the compoundof formula VIII, and

FIG. 5 shows the proton NMR spectrum of the compound of figure VIII inCDCl₃.

EXAMPLES Example 1

Characterization of strain F/94-499709 (DSM 10275)

The following medium is used to characterise the strain F/94-499709, inwhich the media components are given as weight/volume in de-ionizedwater and heat sterilization is performed for 20 minutes at 121° C.

MEA: 2% malt extract, 0.4% yeast extract, 2% agar.

The strain F/94-499709 shows the following characteristics whenpoint-inoculated on MEA in Petri dishes and incubated in the dark:

The optimal temperature for growth is between 24 and 30° C. After 14days of incubation colonies attain a diameter of 25 to 32 mm at 24° C.,30 to 37 mm at 27° C. and 7 to 15 mm at 33° C. Above 37° C. and below13° C. strain F/94-499709 does not show any growth.

Colonies growing at 27° C. in the dark are generally cream colored tolight buff, remain rather flat to slightly raised, with little andrestricted whitish to light gray aerial mycelium developing in thecenter. Radial furrows can become conspicuous, and when viewed from thebottom, darker gray concentric zones can become predominant. In agingcultures the aerial and substrate mycelium in the central parts of thecolonies can become dark gray, whereas the colony edges remain creamcolored to light buff.

Upon microscopic examination no sporulating structures have beenobserved and thus strain F/94-499709 is tentatively termed a myceliumsterilum.

Example 2

Fermentation of strain F/94-499709.

The following media and procedures are suitable for use in a process ofproducing the compound of formula VIII by fermentation of the strainF/94-499709. If not otherwise stated, all media components are given asweight/volume in de-ionized water and heat sterilization is performedfor 20 minutes at 121° C.

PCM (pre-culture and intermediate culture medium): 2% malt extract, 0.4%yeast extract, 0.1% agar.

PRM (production medium): 2% soluble starch, 0,5% yeast extract, 2%glucose, 2% corn steep liquor, 0,5% peptone, 0,2% calcium carbonate.

Pre-cultures are produced by thawing two ml of a liquid nitrogen seedingsuspension of strain F/94-499709, inoculating them into a 500ml-Erlenmeyer flask containing 200 ml PCM, and incubating it at 24° C.for seven days on a rotary shaker at 200 RPM.

For the production of the primary intermediate culture fourteen 500ml-Erlenmeyer flasks each containing 200 ml PCM are inoculated each withfive ml of the pre-culture.

Secondary intermediate cultures are produced by inoculating two 50 Literfermentors containing PCM each with 1.4 Liters of primary intermediatecultures. The fermentation was carried out for six days under thefollowing conditions: 24° C., 1 Liter air/minute/Liter medium, bladestirrers rotating at 150 RPM and 0.5 bar pressure. For the production ofthe compound of formula VIII and related compounds 13 Liters of thesecondary intermediate culture were inoculated into each of three 500Liter fermentors containing PRM. The fermentation was carried out underthe following conditions: 21° C., 1 Liter air/minute/Liter medium, bladestirrers rotating first at 100 RMP and gradually increasing to 150 RPMand 0.5 bar pressure. 1500 Liters of the production fermentation wereharvested and combined after 96 hours for recovery of desired compoundof formula VIII and related compounds.

Example 3

Isolation of the peptolide of formula VIII from the strain F/94-499709.

The broth from the 1500 liters fermentation together with 1700 liters ofethyl acetate is homogenised in a Dispax reactor and stirred vigorouslyfor 3 hours. The organic phase is separated with the aid of a Westfaliaseparator. This extraction step is repeated and the organic phasesevaporated together under reduced pressure to give 2745 g of extract.The extract is defatted by a three step extraction with 40 liters ofmethanol/water mixture (9:1) and 40 liters of cyclohexane. The methanolfractions are combined and evaporated to dryness under reduced pressureto yield 960 g of defatted extract. This extract is chromatographed on acolumn of 15 kg of Sephadex LH20 in methanol solution to give fractionstotalling 135 g containing the peptolide of formula VIII. 300 g ofSilicagel are impregnated with this 135 g fraction total and theimpregnated Silicagel is then added on to the top of a column of 1.5 kgof Silicagel Merck 0.04 to 0.063 mm and chromatographed with 1 litermethyl-tertiarybutylether/cyclohexane 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3,8:2, 9:1, 3 liters MTBE and 3 liters of MTE/methanol 95:5.

Fractions of 1 liter are collected and analysed by HPLC and TLC.Fractions 8 and 9 are combined and evaporated to dryness.Crystallisation from ether yields 21.9 g of pure peptolide of formulaVIII. Further purification of the mother liquor and fractions 10 to 13by chromatography on Silicagel H Merck (750 g) in a similar way asdescribed above yields a further batch of crystalline cyclopeptolide offormula VIII. The peptolide is found to have a melting point (mp) of143-146° C. when purified from ether and an optical rotation [α]_(D) ²⁰=-233.9°(c=0.908, Methanol). The peptolide of formula VIII, has an IC₅₀of about 2 nM when tested in the VCAM-1 cell ELISA.

Molecular formula: C₅₁ H₈₃ N₇ O₉ (938.3) UV-Spectrum in methanol: γmax(ε')=290 (5.4), 279 (5.08), 220 (38.1), 197 (55.7).--see FIG. 1 IR (KBr)spectrum given in FIG. 2 MS FAB spectrum given in FIG. 3 MS FAB spectrum(with LiI) given in FIG. 4 proton NMR spectrum in CDCl₃ given in FIG. 5MTBE=methyl tertiary butyl ether VCAM=vascular adhesion molecule

Example 4

Synthesis of the N1'-Desmethoxy derivative of the the compound offormula VIII

A solution of 4.9 mg of the compound of formula VIII is dissolved in 3ml methanol and 8 mg of palladium on charcoal (10%) is added. Themixture is stirred under an atmosphere of hydrogen for 2 hours, flushedwith argon, filtered and evaporated to yield the title compound ascolourless foam. The compound is analysed by thin layer chromatographyand NMR spectroscopy and the following results are obtained.

TLC: silica gel, toluene/methanol 9/1, Rf=0.28. 1H-NMR (3 conformers56:37:7, marked with *°', characteristic signals given): 8.72* (d, J=10Hz, NH); 8.08* (s, br, indole NH); 6.97*, 6.90° (2d, J=2 Hz, indoleH-2); 6.34° (d, J=9.5 Hz, NH); 6.00* (d, J=6.5 Hz, NH); 5.83' (d, NH);5.32° (ddd, PrLeu alpha-H); 5.12°, 5.08* (2q, J=7 Hz, lactic acid Me);4.50* (dd, MeLeu alpha-H); 4.10* (ddd, Leu alpha-H); 3.42 (q, J=7 Hz,MeAla alpha-H); 3.43°, 3.19°, 3.12*, 2.93*, 2.53*, 2.35° (6s, NMe);1.54*, 1.5°° (2d, J=7 Hz, MeAla alpha-H), 1.38°, 1.24* (2d, J=7 Hz,lactic acid alpha-H); 0.57*, -0.01* (2d, J=6.5 Hz, Me), -0.19* (ddd, Leubeta-H).

Example 5

Synthesis of the N1'-Methyl derivative of the compound of formula VIII

A solution of 5 mg of the product of example 4 in 0.5 ml dry DMF ismixed with 100 ml iodomethane and a solution of 3 mg of sodiumbis(trimethylsilyl)amide in 0.3 ml DMF is added. After stirring of thereaction mixture for 1.5 h at RT, the mixture is poured onto 0.1 Maqueous HCl, extracted with ethyl acetate and partitioned between ethylacetate and saturated aqueous bicarbonate solution. The organic phase iswashed with brine, dried over sodium sulfate and evaporated in vacuo.The crude product is purified by chromatography on silica gel (gradient:toluene/methanol=100/0.25 to 100/2.5) to yield the title compound as acolorless foam. The compound is analysed by thin layer chromatographyand NMR spectroscopy and the following results are obtained.

TLC: silica gel, toluene/methanol 9/1, Rf=0.40. 1H-NMR (2 conformers60:40, marked with * °, characteristic signals given): 8.72* (d, J=10Hz, NH); 6.79*, 6.74° (2s, indole H-2); 6.35° (d, J=9.5 Hz, NH); 5.98*(d, J=6.5 Hz, NH); 5.32° (ddd, PrLeu alpha-H); 5.12°, 5.08* (2q, J=7 Hz,lactic acid Me); 4.50* (dd, MeLeu alpha-H); 4.06* (ddd, Leu alpha-H);3.73 (s, indiole NMe); 3.44°, 3.19°, 3.15*, 2.93*, 2.53*, 2.35° (6s,NMe); 1.55*, 1.51° (2d, J=7 Hz, MeAla alpha-H), 1.39°, 1.24* (2d, J=7Hz, lactic acid alpha-H); 0.57*, -0.09* (2d, J=6.5 Hz, Me), -0.32* (ddd,Leu beta-H).

Example 6

5-[8,11-Diisobutyl-14-(1-methoxy-1H-indol-3-ylmethyl)-7,13,19,20-tetramethyl-5,17-bis-(2-methyl-hexyl)-3,6,9,12,15,18,21-heptaoxo-1-oxa-4,7,10,13,16,19-hexaaza-cycloheneicos-2-yl]-pent-2-enoicacid methyl ester

A solution of 185 mg of the compound of formula XV (see below) and 1.26g of methoxy-carbonylmethylenetriphenylphosphorane in toluene is stirredat room termperature for 1 hour. Then the solvent is evaporated invacuo, the residue chromatographed on a LH-20 gel-filtration column inmethanol, the product containing fractions are evaporated in vacuo andfurther purified by chromatography on silica gel (eluent: gradienttoluene/methanol=99.5/0.5 to 96/4) to yield the title product as acolorless solid foam. The product is lyophilized from benzene.

¹ H-NMR (CDCl₃, characteristic signals, 3 conformers 53:41:6, markedwith * °'): 8.67* (d, J=10 Hz, C₉ AA NH); 7.87* (d, J=10 Hz, C₉ AA NH);7.80° (d, J=10 Hz, NH); 7.69° (d, J=10 Hz, NH); 7.45-7.35 (4d, MeMeOTrpH-4', H-7'); 7.23*, 7.22° (2m, MeMeOTrp, H-6'); 7.4 (2m, MeMeOTrp H-5');7.06*, 7.00° (2s, MeMeOTrp H-2'); 6.88° (ddd, J=15 Hz, J=7 Hz, --CH═);6.76* (ddd, J=15 Hz, J=7 Hz, --CH═); 6.240 (d, J=10 Hz, Leu NH); 6.04*(d, J=6 Hz, Leu NH); 5.820, 5.78* (2d, J=15 Hz, ═CH--CO); 5.29° (ddd,MeAla a-H); 5.07-4.98 (m, a-H); 4.92 (dd, MeMeOTrp a-H); 4.86* (ddd, C₉AA a-H) 4.78* (dd, Leu a-H); 4.71 (m, a-H); 4.48* (dd, MeLeu a-H); 4.17*(ddd, Leu a-H); 4.06*, 4.03', 4.02° (3s, N-OMe); 3.75*, 3.74° (2s,COOMe); 3.43° (s, N-Me); 3.20*(s, MeAla NMe); 3.17°(s, N-Me); 2.92* (s,MeMeOTrp N-Me); 2.52* (s, MeLeu N-Me); 2.47° (s, N-Me); 1.51*, 1.480(2d, J=7 Hz, MeAla β-Me); 1.04 (d, J=6.5 Hz, MeLeu Me); 0.98-0.84 (m);0.63* (d, J=6.6 Hz, Leu Me); 0.56', 0.39' (2d); 0.06* (d, J=6.6 Hz, LeuMe); -0.11* (ddd, Leu β-CH).

Analogously as described in example 6 the ethyl ester is obtained.

The starting material of formula XV ##STR13## is known (WO 96/03430). Inthis formula the substituents have the following significances:##STR14## In the following examples 7-11, the same abbreviations areused with the exception that: ##STR15##

Example 7

Compound of formula IX

(i.e. the compound of formula I, in which A=A", R₈ =thiazol-2-yl, B=B',R₁ =CH₃, C=C", R₃ =OCH₃, ═=db, X=X',Y=Y')

A solution of 400 mg of the compound of formula I in which A=A", R₈=--CS NH₂, B=B' R₁ =CH₃, C=C", R₃ =OCH₃, ═=db, X=X', Y=Y') and 0.5 ml ofchoroacetaldehyde hydrate in 8 ml of dry dimethylformamide is stirredwith 0.5 g 4 Å molecular sieve and heated to 600 for 5 hours. Themixture is then diluted with ethyl acetate, filtered and extracted with0.1 N HCl. The organic phase is washed with brine, dried and evaporatedin vacuo. The crude product is purified by chromatography on silica gel(eluent: gradient toluene/methanol=99.5/0.5 to 98/2) to yield the titlecompound as a solid foam.

Analogously as described in example 7 the following compounds of formulaI are obtained (A=A", B=B' R₁ =CH₃,C=C",R₃ =OCH₃, ═=db, X=X',Y=Y')

    ______________________________________                                        Example   R.sub.8                                                             ______________________________________                                         8                                                                                       ##STR16##        solid foam                                         9                                                                                       ##STR17##        "                                                 10                                                                                       ##STR18##        "                                                 11                                                                                       ##STR19##        "                                                 ______________________________________                                    

The starting material, i.e. the compound of formula I in which A=A', R₈=--CS NH₂, B=B' R₁ =CH₃, C=C', R₄ =OCH₃, ═=db, X=X', Y=Y', is preparedin the following manner:

A solution of 1.1 g of the compound of formula XI (A is an α hydroxysubstituted butyric acid residue γ substituted by --CN B=B' R₁ =CH₃,C=C', R₄ =OCH₃, ═=db, X=X', Y=Y') and 1.8 g of diphenylphospinodithioicacid in 25 ml of isopropanol is heated to reflux for 7 hours. Thereaction mixture is evaporated in vacuo, dissolved in ethyl acetate andextracted with sodium bicarbonate solution and brine. After evaporationof the organic layer the crude product is purified by chromatography onsilica gel (eluent: gradient toluene/methanol=99.5/0.5 to 98/2) to yieldthe starting compound as colorless foam.

The compounds of Examples 4 to 11 have activities similar to that of thecompound of formula VIII when measured in the VCAM-1 cell ELISA.

¹ H-NMR Spectra (CDCl₃)

Ex.

7. 3 conformers 46:51:3, marked with *°': 8.70* (d, J=10 Hz, LeuPr NH);7.89* (d, 10 Hz, NH); 7.83° (d, J=9 Hz, NH); 7.69, 7.66 (2d, J=3.3 Hz,thiazole H); 7.58 (d, J=10 Hz, NH); 7.53*, 7.47° (2dm, J=7 Hz, MeTrpOMeH-4'); 7.38*, 7.37° (2dm, J=8 Hz, MeTrpOMeH-7'); 7.22*, 7.20° (2tm,MeTrpOMe, H-6'); 7.17, 7.16 (2d, J=3.3 Hz, thiazole-H); 7.09 (s,MeTrpOMe H-2'); 7.03*, 7.00° (2dd, MeTrpOMe H-5'); 6.98° (s, MeTrpOMeH-2'); 6.18° (d, J=10 Hz, Leu NH); 6.03* (d, J=7 Hz, Leu NH); 5.80' (d,J=10 Hz, Leu NH); 5.30° (ddd, LeuPr a-H); 5.11* (dd, hydroxybutyric acida-H); 5.05-4.98 (m, a-H); 4.94 (dd, MeTrpOMe a-H); 4.86° (ddd, LeuPra-H); 4.73 (m, a-H); 4.49* (dd, MeLeu a-H); 4.23* (ddd, Leu a-H); 4.02,4.00 (2s, N-OMe); 3.84° (m, a-H); 3.59-3.40 (m); 3.38 (q, J=7 Hz, MeAlaa-H); 3.33 (s, N-Me); 3.2-2.85 (m); 3.21 (s, N-Me); 3.07 (s, N-Me);2.93* (s, MeTrpOMe N-Me); 2.53* (s, MeLeu N-Me); 2.49 (s, N-Me);2.43-2.28 (m); 2.18 (m); 1.98 (m); 1.81 (m); 1.7-1.1 (m); 1.48, 1.46(2d, J=7 Hz, MeAla β-Me); 1.06* (d, J=6.5 Hz, MeLeu Me); 1.00-0.84 (m);0.61* (d, J=6.6 Hz, Leu Me); 0.54' (d, J=6.6 Hz, Leu Me); 0.35' (d,J=6.6 Hz, Leu Me); 0.10* (d, J=6.6 Hz, Leu Me); -0.12* (ddd, Leu β-CH).

8. 3 conformers 47:48:5, marked with *°': 8.64* (d, J=10 Hz, LeuPr NH);7.82 (2d, 10 Hz, NH); 7.63° (d, J=10 Hz, NH); 7.23, 7.17, 7.08, 6.97(4t, arom. H); 7.32, 7.28 (2s, thiazole-H); 7.07 (s, MeTrpOMe H-2');6.81 (s, MeTrpOMe H-2'); 6.220 (d, J=10 Hz, Leu NH);

6.07* (d, J=7 Hz, Leu NH); 5.80' (d, J=10 Hz, Leu NH); 5.29° (ddd, LeuPra-H); 5.20* (dd, MeTrpOMe a-H); 5.12° (dd, hydroxybutyric acid a-H);5.030 (ddd, LeuPr a-H); 4.97* (ddd, LeuPr a-H); 4.85 (m, hydroxybutyricacid+LeuPr a-H); 4.710 (ddd, Leu a-H); 4.52* (dd, MeLeu a-H); 4.21*(ddd, Leu a-H); 4.02, 3.90 (2s, N-OMe); 3.34 (s, N-Me); 3.22 (s, N-Me);3.02 (s, N-Me); 2.93 (s, N-Me); 2.53 (s, N-Me); 2.48 (s, N-Me); 1.43,1.42 (2d, J=7 Hz, MeAla β-Me); 1.06* (d, J=6.5 Hz, MeLeu Me); 0.62* (d,J=6.6 Hz, Leu Me); 0.10* (d, J=6.6 Hz, Leu Me); -0.04* (ddd, Leu β-CH).

9. 3 conformers 42:52:6, marked with *°': 8.67* (d, J=10 Hz, LeuPr NH);7.83, 7.80 (2d, 10 Hz, NH); 7.63° (d, J=10 Hz, NH); 7.55, 7.42, 7.39,7.36 (4d, MeTrpOMe arom.); 7.22, 7.18, 7.05, 6.97 (4dd, MeTrpOMe H-5',H-6'); 7.06° (s, MeTrpOMe H-2'); 6.88* (s, MeTrpOMe H-2'); 6.220 (d,J=10 Hz, Leu NH); 6.02* (d, J=7 Hz, Leu NH); 5.77' (d, J=10 Hz, Leu NH);5.30° (ddd, LeuPr a-H); 5.21* (dd, MeTrpOMe a-H); 5.0 (m, a-H); 4.85 (m,a-H); 4.65 (ddd, Leu a-H); 4.49* (dd, MeLeu a-H); 4.13* (ddd, Leu a-H);4.03, 3.98 (2s, N-OMe); 3.70 (m); 3.55 (m); 3.45 (q, J=7 Hz, MeAla a-H);3.37, 3.20, 3.11, 2.93, 2.52, 2.43 (6s, N-Me); 2.73 (m,tetrahydro-benzothiazol); 1.50, 1.48 (2d, J=7 Hz, MeAla β-Me); 1.04* (d,J=6.5 Hz, MeLeu Me); 0.58* (d, J=6.6 Hz, Leu Me); 0.50' (d, J=6.6 Hz,Leu Me); 0.30' (d, J=6.6 Hz, Leu Me); 0.03* (d, J=6.6 Hz, Leu Me);-0.22* (ddd, Leu β-CH).

10. 3 conformers 44:51:5, marked with *°': 8.66* (d, J=10 Hz, LeuPr NH);7.83, 7.81 (2d, 10 Hz, NH); 7.630 (d, J=10 Hz, NH); 7.57*, 7.43°, 7.38*,7.36° (4d, J=8 Hz, indole-H); 7.21*, 7.18°, 7.05, 6.97 (4t, indole-H);7.06* (s, MeTrpOMe H-2'); 6.88° (s, MeTrpOMe H-2'); 6.70°, 6.69* (2q,J=1 Hz, thiazole-H); 6.23° (d, J=10 Hz, Leu NH); 6.05* (d, J=7 Hz, LeuNH); 5.80' (d, J=10 Hz, Leu NH); 5.29° (ddd, LeuPr a-H); 5.11° (dd,hydroxybutyric acid a-H); 5.01 (m); 4.97 (dd, a-H); 4.85 (m, 2×a-H);4.69° (ddd, Leu a-H); 4.57' (dd, a-H); 4.48* (dd, MeLeu a-H); 4.16*(ddd, Leu a-H); 4.03, 3.98 (2s, N-OMe); 3.43 (q, J=7 Hz, MeAla a-H);3.37, 3.20, 3.10, 2.92, 2.52, 2.46 (6s, N-Me); 2.40, 2.39 (2d, J=1 Hz,Me-thiazole); 1.48, 1.47 (2d, J=7 Hz, MeAla β-Me); 1.05* (d, J=6.5 Hz,MeLeu d-Me); 0.60* (d, J=6.6 Hz, Leu d-Me); 0.52', 0.33' (2d, J=6.5 HzLeu d-Me); 0.05* (d, J=6.6 Hz, Leu d-Me); -0.14* (ddd, Leu β-CH).

11. 3 conformers 47:50:3, marked with *°': 8.69* (d, J=10 Hz, LeuPr NH);7.79, 7.78 (2d, 10 Hz, NH); 7.650 (d, J=10 Hz, NH); 7.51*, 7.41°, 7.39*,7.38° (4d, J=8 Hz, indole-H); 7.23*, 7.20°, 7.07, 7.00 (4t, indole-H);7.04* (s, MeTipOMe H-2'); 6.85° (s, MeTrpOMe H-2'); 6.71°* (s,thiazole-H); 6.25° (d, J=10 Hz, Leu NH); 6.04* (d, J=7 Hz, Leu NH);5.80' (d, J=10 Hz, Leu NH); 5.30° (ddd, LeuPr a-H); 5.10° (dd,hydroxybutyric acid a-H); 5.02 (m); 4.97 (dd, a-H); 4.85 (m, 2×a-H);4.72° (ddd, Leu a-H); 4.60' (dd, a-H); 4.50* (dd, MeLeu a-H); 4.17*(ddd, Leu a-H); 4.04,4.00 (2s, N-OMe); 3.48 (q, J=7 Hz, MeAla a-H);3.41, 3.21, 3.17, 2.92, 2.53, 2.47 (6s, N-Me); 0.97, 0.96 (2s,t-Bu-thiazole); 1.50, 1.49 (2d, J=7 Hz, MeAla β-Me); 1.06* (d, J=6.5 Hz,MeLeu d-Me); 0.62* (d, J=6.6 Hz, Leu d-Me); 0.53', 0.35' (2d, J=6.5 HzLeu d-Me); 0.07* (d, J=6.6 Hz, Leu d-Me); -0.08* (ddd, Leu β-CH).

A) 3 conformers 44:30:26, marked with *°': 8.85 (d, CSNH₂); 8.60 (d,LeuPr NH); 8.17 (d, CSNH₂); 8.03, 8.00 (2d, NH); 7.88 (m, CSNH₂);7.6-7.1 (arom.); 6.28* (d, 10 Hz, Leu NH); 6.07° (d, 7 Hz, Leu NH);5.87' (d, 9 Hz, Leu NH); 5.26* (ddd, LeuPr a-H), 5.22 (dd, hydroxy acida-H); 5.15-4.95, 5.08* (dd, hydroxy acid a-H); 4.83° (ddd, LeuPr a-H);4.50* (ddd, Leu a-H); 4.37* (dd, MeTrpOMe a-H); 4.25° (ddd, Leu a-H);4.09, 4.05, 4.03* (3s, OMe); 3.89 (m, a-H); 3.65*, 3.63', 3.52° (3q, 7Hz, MeAla a-H); 3.57 (m), 3.17, 3.16, 3.15, 3.22, 3.20, 3.05, 2.92,2.55, 2.53 (9s, N-Me); 1.8-1.1; 1.05 (d, 7 Hz); 0.99-0.82, 0.60°, 0.55',0.23', 0.17° (4d, 7 Hz, Leu d-Me); -0.15°, -0.17' (ddd, Leu b-CH). PKF285-916 (thiazole).

Biological Activity

The activities of the compounds of the invention are tested in assaysfor cytotoxicity and inhibition of ICAM-1, VCAM-1 and E-selectinexpression, cell proliferation, as well as for inhibition of TNF releaseand a corresponding assay for cytotoxicity.

The assays are carried out as follows:

HaCaT cells, a spontaneously-transformed, non-tumorigenic humankeratinocyte cell line with highly preserved phenotypic differentiationcharacteristics of normal keratinocytes (Boukamp et al., 1988 J. CellBiol. 106, 761-771), are used both for the cell proliferation assay andthe ICAM-1 cell Elisa.

A. ICAM-1 CELL-ELISA ASSAY

I. Keratinocyte ICAM-1 Cell Elisa

The ICAM-1 cell Elisa used to determine inhibition of ICAM-1 expressionis substantially as described by Winiski and Foster (1992, J. Invest.Dermatol., 99, 48-52). HaCaT cells are seeded in 96 well microtiterplates (2×10⁴ cells/well in culture medium: DMEM with 5% FCS, 100 U/mlPenicillin, 100 mg/ml Streptomycin, 2 mM Glutamine, 1 mM Na Pyruvate),grown to confluency, and then incubated in fresh test medium (as forculture medium but with 0.5% FCS instead of 5%) with or withoutIFN-γ/TNF-α stimulation medium (test medium+1000 U/ml IFN-γ/3 ng/mlTNF-α) both in the presence and absence of the test compound for ca. 24hrs. The medium is then washed away and the cell monolayers are fixedwith 1% parafomraldehyde. The monolayers are incubated with saturatingamounts of primary (mouse anti-ICAM-1 monoclonal) and secondary (goatanti-mouse peroxidase conjugated) antibodies. The subsequent peroxidasereaction uses 3-amino-9-ethylcarbazole (AEC) as substrate and generatesan insoluble, colored product, which is easily measured in a standardmicrotiter plate reader.

II. Measure of Cytotoxicity

After the AEC reaction to detect ICAM-1 is completed, the HaCaTmonolayers, are rinsed with PBS (200 mL), the PBS is poured off from theplates which are then patted dry on top of a paper towel to removeexcess liquid. The bottom surfaces of the microtitre plates are gentlywiped with a moist facial tissue and then again with a dry facial tissueand absorbance read at 492 nm. Before the monolayers can dry out, 0.1 mlof 0.1% crystal violet solution in PBS (passed first through a 0.2 mmfilter) is added to each well. The plates are then incubated at roomtemperature for 10 minutes, washed thoroughly 5× with PBS, excess fluidremoved as described above and their absorbance read again at 492 nmbefore the monolayers are able to dry out. Subtraction of opticaldensities before and after staining gives values due to crystal violetstaining and is hence related to the amounts of cell monolayer presentin the wells. These values are used to correct the AEC values.

B. Endothelial cell VCAM-1, ICAM-1 and E-selectin Cell-Elisa Assay

The assay is based on a 96-well cell Elisa method using the humanmicrovascular endothelial cell line HMEC-1 and human umbilical veinendothelial cells (HUVEC). Cells are pretreated for four hours with thetest compound, stimulated for the next 6-16 hours with TNFα, thenparafomaldehyde-fixed for subsequent evaluation of VCAM-1, ICAM-1 orE-selectin expression by an indirect immunoperoxidase stainingtechnique. Cytotoxic effects are determined by counting the relativenumber of cells (Giemsa nuclear stain) after exposure to the testsubstances, in comparison to the control wells (solvent and media only).Compounds are scored positive if they exhibit >50% VCAM-1, ICAM-1 orE-selectin inhibition with <25% cell loss.

Methodology

I. Cell line: The VCAM-1 and ICAM-1 assay utilizes an immortalized(SV-40 virus large T antigen) human microvascular endothelial cell line(HMEC-1; Ades et al., J. Invest. Dermatol. 99: 683-690, 1992). HMEC-1cells constitutively express low levels of ICAM-1 which are upregulatedby inflammatory mediators. However, they only express VCAM-1 followingcytokine stimulation. Dose-response and time-course experiments wereperformed to determine the optimal conditions for inducing VCAM-1 andICAM-1 expression.

II. Growth conditions: HMEC-1 cells are grown in T-75 flasks (Nunc)under standard conditions (37° C., 5% C02) with 1.5×10⁶ cells/ml culturemedium (CM=Endothelial Cell Basal Medium [EBM; Clonetics] supplementedwith 10% FCS, 10 ng/ml human EGF (Boehringer), 1 mg/ml hydrocortisone(Sigma #0888), 2.2 g/l NaHCO₃, 15 mM Hepes, 0.11 g/l sodium pyruvate, 4mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin). Aftermild trypsinization (0.25% trypsin+0.1% EDTA for 8 min) andresuspension, the cells are reseeded every 2-3 days at a 1:3 splittingratio.

III. VCAM-1 and ICAM-1 Cell-Elisa

96 well flat-bottom microtiter plates are precoated with bovinefibronectin (FN; Sigma #F1141) and then seeded with 2×10⁴ cells/well in200 ml of EBM growth medium and incubated overnight. The following daythe culture medium (CM) is initially replaced with 2.00 ml/well of EBMassay medium (CM supplemented with 5% FCS instead of 10%) andsubsequently replaced with 180 ml of medium containing either (1)appropriate concentrations of the test compound, (2) correspondingconcentrations of solvent/methanol-extracted medium, or (3) EBM assaymedium alone and incubated for 4 hr at 37° C. Each 96-well assay isperformed with duplicate wells. The cells are then stimulated by adding20 ml of concentrated cytokine solution (2000 U/ml TNFa) and incubatedfor 16 hr at 37° C.

The cell monolayer is then washed with 1% parafornaldehyde in EBMmedium, fixed in 2% parafomaldehyde for 15 min at room temperature (RT)and rinsed several times with PBS. The PBS is removed from the cells,and the monolayer is incubated for 30 min in PBS containing 10% normalgoat serum (NGS). The NGS solution is replaced with 100 ml/well of theanti-VCAM-1 or ICAM-1 monoclonal antibody and incubated overnight at 4°C. The mAb solution is then removed and the cells rinsed several timeswith PBS, followed by incubation with PBS containing 10% NGS for 30-60min at RT. The NGS solution is removed and 100 ml of horseradishperoxidase-conjugated goat F(Ab')₂ anti-mouse IgG antibody (Tago; 1:500dilution in PBS containing 5% NGS) is added and the plates incubated for1 hr at RT. The secondary antibody is then removed and the cells rinsedin PBS, which is then replaced with 150 mn/well of a freshly-preparedand filtered AEC solution (3-amino-9ethyl-carbazole; Sigma) and theplates incubated for 45-60 min at RT. The peroxidase substrate isremoved and the cells rinsed in PBS. AEC absorbance values are read on amicrotiter plate reader at 550 nm and corrected for "blank" or referencevalues at 690 nm.

IV. E-selectin assay: The E-selectin assay is performed using freshlyisolated HUVEC, essentially as described for the VCAM-1 or ICAM-1 assayexcept for a shorter TNFa-stimulation (6-8 hours).

V. Measure of Cytotoxicity (Cell loss based on nuclear stain):

The endothelial cells are destained by replacing the PBS with 95%ethanol for 20 min (two 10 min changes) with control by microscopicevaluation. The cells are then rinsed in distilled water (Aquadest) andthe monolayer covered with a 33% Giemsa solution in Aquadest for 5 minat RT. The wells are then washed with Aquadest and air dry for at least15 min. Microscopic evaluation is used to check that only the nuclei arestained, with essentailly no cytoplasmic staining. Giemsa absorbancevalues are read on a microtiter plate reader at 550 nm and corrected for"blank" values (rows without cells) at 690 nm.

VI. Data Evaluation: The AEC values for constitutive VCAM-1 orE-selectin expression (unstimulated control wells) are essentially equalto those of an isotype-matched control mAb and represent the backgroundstain. In every 96-well plate, the mean constitutive value is subtractedfrom the mean AEC value for each cytokine-stimulated group (EBM andsolvent controls, as well as test substance), resulting in a numberwhich represents upregulated ICAM-1 and inducible VCAM-1 or E-selectinCell adhesion molecule (CAM) expression (referred to as AEC-CAM). EachAEC-CAM value is then divided by the corresponding mean Giemsa value,resulting in a number which estimates relative levels of CAM expressionfor a given cell density, based on the number of nuclei (referred to asAEC: Giemsa ratio).

    AEC(stimulated)-AEC(unstimulated)=AEC-CAM

    AEC-CAM/Giemsa=AEC:Giemsa ratio

Therefore "actual" CAM IC₅₀ values are determined by comparing theAEC:Giemsa values for a test substance with those of the stimulatedcontrol (EBM, solvent). These values are then analyzed relative to theIC₅₀ values for Giemsa alone. Strict criteria determine whether the CAMinhibition versus cytotoxicity (Giemsa) profile indicates a "real" hitwhich should be pursued.

C. HaCaT cell PROLIFERATION ASSAY

HaCaT cells are cultivated in DMEM (Gibco #074-02100) supplemented with2.2 g/l NaHCO₃, 0.11 g/l sodium pyruvate, 15 mM Hepes, 5% fetal calfserum (FCS), penicillin (100 U/ml), streptomycin (100 mg/ml), andglutamine (to increase the final concentration by 4 mM). For theproliferation assay, cells are detached by trypsinization, suspended infresh medium, and seeded into 96-well microtiter plates at a finaldensity of 4000 cells/0.2 ml/well. After 24 hours (day 0) the medium isreplaced with fresh medium containing graded concentrations of testcompound. After 3 days of incubation at 37° C./5%CO₂, the extent ofcellular proliferation in comparison to solvent controls is measured bya colorimetric assay that measures relative cell mass using the dyesulforhodamine B (Skehan et al, 1990, J. Natl. Cancer Inst. 82,1107-1112). The "starting cell number" is determined by measuring therelative cell mass on day 0. The results are expressed as %Inhibition=100-% control absorbance (where solvent control=100%) andrepresent the average±standard deviation of three measurements. Adose-response curve is plotted semi-logarithmically and theconcentration required for half-maximal inhibition (IC₅₀) is determinedby linear interpolation. Maximal inhibition without net loss of cells isrepresented by the "starting cell number" and is usually between 90-98%.

D. Inhibition of TNF release

I.

Mononuclear cells are prepared from the peripheral blood of healthyvolunteers using ficoll-hypaque density separation according to themethod of Hansell et al. (J. Imm. Methods (1991) 145: 105.) and used ata concentration of 10⁵ cells/well in RPMI 1640 plus 10% FCS. Cells areincubated with serial dilutions of the test compounds for 30 minutes at37° C. prior to the addition of IFNγ (100 U/ml) and LPS (5 mg/ ml) andsubsequently further incubated for three hours. Incubation is terminatedby centrifugation at 1400 RPM for 10 min. TNFα in the supernatant ismeasured using a commercial ELISA (Innotest hTNFα, available fromInnogenetics N.V., Zwijnaarde, Belgium). The compounds are tested atconcentrations of from 0 to 10 mM. Exemplified compounds of formula I,especially preferred compounds of formula Ip, Ip', Ip", VII, IX and X,suppress TNF release in this assay with an IC₅₀ of from about 5 up toabout nM.

II. Cytotoxicity

Cytotoxicity is determined on THP1 cells (5×10⁴ /well) which areincubated in the presence of IFNγ(100 U/ml) and LPS (5 mg/ ml) andpresence and absence of test compound for 24 hours at 37° C. Percentagesof living and dead cells are assessed by a colorimetric readout (MTT),which measures mitochondrial dehydrogenase enzymes in living cells, asdescribed in Mosman, J. Imm. Methods (1983) 65:55. Preferred compoundsof the invention typically have comparitively low cytotoxicity whenmeasured in this assay, e.g. an IC₅₀ of from about 100 up to about 1000nM.

We claim:
 1. A cyclopeptide of formula I in free, salt or ester form:##STR20## Wherein A is substituted by: H, methyl, ethyl, propyl, orvinyl, optionally substituted byHalogen, alkoxy, optionally protectedhydroxy or amino, CSNH2, COOR2, vinyl, --C.tbd.CH or thiazole, whereinR2 is H or lower alkyl, optionally substituted byalkyl, halogen,cycloalkyl, optionally substituted thiazole, COOR2 or --C.tbd.CH,wherein R2 is as defined above; R1, R2, R3 are H or methyl; R4 is H,alkoxy, alkyl, or benzyl; R5 is H or halogen; ═ represents a single ordouble bond; P is C2 to C14 alkyl; X is C2to C10 alkyl.
 2. A compound offormula I ##STR21## Wherein A is H, ethyl, or methyl; R1, R2, R3 arehydrogen or methyl;R4 is Cl to C4 alkoxy; R5 is H or halogen; ═represents a single or double bond; P is C2 to C14 alkyl; X is C2 to CIOalkyl.
 3. A compound of claim 1 wherein A is: ##STR22## wherein R6represents a lower alkyl group.
 4. A compound of claim 1, wherein A is:##STR23## Wherein R7 represents hydrogen, lower alkyl, phenyl or forms acarbocyclic ring together with position 5 of the thiazolyl ring.
 5. Acyclopeptolide of formula VII, IX or X in free or salt form ##STR24##